Systole Diastole
6.3. Results
with post hoc testing using the most stringent pairwise Holm-Sidak test.
Statistical significance was considered as p < 0.05.
Fig. 6.1. TGF-β1 synthesis and signaling. (A) 5-HT2B (hatched bar) and AT1 agonists (double hatched bar) lead to increased TGF-β1 synthesis, while both antagonists prevent synthesis induced by either agonist treatment (blue and orangebars). * indicates significant difference (p <
0.005) versus control.
AT1 antagonism does not prevent TGF-β1 activation of AVICs
We have previously shown that 5-HT2B antagonism inhibits TGF-β1- induced activation of AVICs; therefore, we hypothesized that antagonism of AT1, a GPCR similar to 5-HT2B, may also be able to inhibit AVIC activation. To test this hypothesis, AVICs were treated with log doses of either 5-HT2B antagonist (SB204741) or AT1 antagonist (ZD7155) for 30 min prior to the addition of 1 ng/ml TGF-β1. AVICs exhibit significantly increased expression of αSMA following 24 h of the TGF-β1 treatment. Consistent with our previous findings, 1 and 10 µM treatments of SB204741 significantly inhibit TGF-β1-induced AVIC
Relative TGF-1
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BW723c86 Ang II SB204741 ZD7155
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activation in a dose dependent manner. However, ZD7155 treatment is less effective in mitigating the AVIC differentiation (Fig. 6.2).
Fig. 6.2. 5-HT2B and AT1 antagonist effect on TGF-β1-induced activation of AVICs. AVICs treated 1 ng/ml TGF-β1 for 24 exhibit significantly increased αSMA expression. Both 1 and 10 μM 5- HT2B antagonist (SB204741) treatments blocked this activation. The AT1 antagonist (ZD7155) treatments were less effective in inhibiting AVIC activation. * indicates significant difference (p <
0.005) versus control.
Treating AVICs with 1 µM ZD7155 has no effect on AVIC activation by TGF-β1, and while 10 µM ZD7155 significantly decreases increased αSMA expression due to TGF-β1 treatment, these AVICs still exhibit significantly higher αSMA than non-treated control samples. In addition to assaying for AVIC
myofibroblast activation, we were also interested in assessing changes in a functional outcome indicative of CAVD—calcific nodule formation. Our lab has
Relative SMA
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recently developed an in vitro model system to study morphogenesis of dystrophic calcific nodules in vitro [199] that are formed due to an imbalance of forces between TGF-β1 treated contractile myofibroblastic AVICs and externally applied cyclic strain. To test the effects of the antagonist treatments on calcific nodule formation, AVICs were seeded in Bioflex culture plates and treated with either 1 µM or 10 µM of either antagonist for 30 min prior to the addition of 1 ng/ml TGF-β1 for 24 h. Following the 24 h incubation, 15% cyclic strain at 1 Hz was added to the cultured AVICs for an additional 24 h.
Fig. 6.3. 5-HT2B and AT1 antagonist effect on TGF-β1-induced calcific nodule formation. (A) AVICs treated 1 ng/ml TGF-β1 for 24 exhibit robust calcific nodule formation following 15% strain for 24 h. Both 1 and 10 μM 5-HT2B antagonist (SB204741) treatments significantly blocked this nodule formation; however, the AT1 antagonist (ZD7155) treatments did not inhibit calcific nodule morphogenesis. (B) Alizarin red staining was used to identify nodules in all treatment groups. * indicates significant difference (p < 0.005) versus TGF-β1 treated control.
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AVICs treated with TGF-β1 alone formed robust calcific nodules on the surface of the Bioflex plates (Fig. 6.3A). Similar to our previous findings, 5-HT2B antagonism prevented formation of calcific nodules in a dose-dependent manner [211];
however, treating AVICs with ZD7155 did not significantly inhibit TGF-β1-induced calcific nodule formation as identified by Alizarin red staining (Fig. 6.3B).
AT1 antagonism inhibits canonical, but not non-canonical, TGF-β1 signaling
Given the divergence of 5-HT2B and AT1 antagonism in inhibiting TGF-β1- induced activation of AVICs, we were next interested in understanding the effect of these antagonist treatments on TGF-β1 signaling. We have previously demonstrated that 5-HT2B antagonism inhibits TGF-β1 induced phosphorylation of p38, but does not inhibit canonical Smad3 activation [211]. In this study, we assessed the phosphorylation state of both of these signaling proteins after 1 h of TGF-β1 treatment and treatment with each antagonist (Fig. 6.4A).
Fig. 6.4. 5-HT2B and AT1 antagonist effect on TGF-β1 canonical and non-canonical signaling. (A) AVICs treated 1 ng/ml TGF-β1 for 1h exhibit increased phosphorylation of both Smad3 (pSmad3) and p38 (pp38). The 5-HT2B antagonist (SB204741) treatments does not effect pSmad3;
however, pp38 was inhibits in a dose dependent manner. In contrast, AT1 antagonist treatment (ZD7155) significantly inhibits pSmad3, but does not significantly inhibit pp38. (B) A PAI-1 reporter plasmid supports the pSmad3 results as both ZD7155 treatments lead to decreased reporter activity. * indicates significant difference (p < 0.005) versus TGF-β1 treated control.
Consistent with our previous results, 5-HT2B antagonism inhibits TGF-β1- mediated phosphorylation of p38 in a dose dependent manner; however, AT1
antagonism does not prevent activation of this non-canonical pathway.
Interestingly, however, AT1 antagonism significantly inhibits TGF-β1-induced phosphorylation of Smad3. To further assess canonical TGF-β1 signaling activity AVICs were transfected to express a partial promoter sequence of PAI-1 (p3TP- lux), a common transcriptional target of activated Smad3, with a luciferase reporter gene. Similar to the Smad3 phosphorylation results, AT1 antagonism significantly inhibits TGF-β1-induced PAI-1 promoter activity in a dose dependent manner with less activity observed at both 1 µM and 10 µM AT1 antagonist treatment.
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p38 MAPK signaling is required for TGF-β1 activation of AVICs
To further assess the importance of p38 phosphorylation in TGF-β1 signaling, AVICs were transfected with a plasmid to express a dominant negative form of p38 that was mutated to remove kinase activity and prevent subsequent downstream signaling. Control AVICs transfected with the plasmid backbone (i.e., missing the dominant negative p38 insert) display a normal two-fold increase in αSMA after treatment with 1 ng/ml TGF-β1 for 24 h; however, the introduction of dominant negative p38 significantly reduces TGF-β1-induced αSMA expression (Fig. 6.5).
Fig. 6.5. Requirement for p38 phosphorylation in AVIC activation by TGF-β1. AVICs treated with TGF-β1 exhibit a significant increase in αSMA; however, AVICs transfected to express a dominant-negative form of p38 (dn p38) do not significantly increase αSMA expression following TGF-β1 treatment. * indicates significant difference (p < 0.005) versus non-treated control.
+TGF-1 Media only
SMA expression
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